ABSTRACT Heart disease is the leading cause of death in the United States, claiming over 600,000 lives annually. The measurement of blood-based biomarkers to narrow differential diagnoses and triage patients plays a critical role in the management of acute and chronic cardiovascular disease. This R01 project will develop a handheld point- of-care test (POCT) that uses low-cost, disposable assay cassettes to measure multiplexed panels of cardiac biomarkers directly from a drop of whole blood. The assay brings together major advances in carbon nanotubes (CNTs) integrated into thin-film transistors (CNT-TFTs) and an assay platform (called ?D4?) for detecting protein analytes with ELISA-like performance from a single drop of blood. Both the CNT-TFTs and D4 are fabricated by printing, making them readily customizable and low cost. CNT-TFTs offer extremely sensitive nanotubes for electrical transduction in an assay and, unlike prior CNT-based biosensors, the CNT-TFTs provide reproducible performance and high yield. The D4 is a fluorescence-based assay that uses antibody (Ab) pairs printed into a polymer brush coating (?POEGMA?) that eliminates background noise and permits highly sensitive measurements directly in complex biological milieu. Merging these two innovative technologies leads to the ?D4- TFT?, which combines the electronic sensitivity of the CNT-TFTs with the nonfouling selectivity of the D4 to yield an assay that can rapidly detect biomarker concentration from whole blood without rinsing, via electrical transduction. Extensive preliminary data demonstrate the D4-TFT?s fully printed fabrication, preservation of transistor operation, and successful detection of target analytes from serum and blood. The specific aims of this project include the development of a handheld POCT unit, with all needed control electronics and a simple user interface with display, and adaptation of the D4-TFT to compatible cassettes that are inserted into the handheld unit for operation. Analytical performance of the D4-TFT assay will be maximized and multiplexed against two panels of cardiac biomarkers: (1) A panel of validated cardiac biomarkers that includes creatine kinase (CK), CKMB, troponin (Tn), and N-terminal pro b-type natriuretic peptide (BNP), which are central to the diagnosis and management of a wide spectrum of cardiovascular conditions, including acute chest pain, myocardial infarction, and heart failure (HF); and (2) two investigative cardiac biomarkers, angiopoietin 2 (ANG2) and tumor necrosis factor alpha (TNF?), which are of major interest for their potential association with bleeding events in advanced HF patients with left ventricular assist device (LVAD) implants (the most common complication in this population). These two diverse panels of cardiac biomarkers will demonstrate the versatility and value of this POCT, both for diagnostic potential using validated biomarkers as well as for rapid clinical screening of investigative biomarkers. Accomplishing these aims will lead to a novel POCT for cardiac biomarkers that can be scaled up easily. Its fully printed nature makes the D4-TFT highly customizable and broadly applicable to other blood-based tests for any biomarker with immunoassay reagents.